Abstract:

Disclosed is an antibody which can recognize BRAK (CXCL14). Also disclosed
is use of the antibody. The antibody can recognize a polypeptide
comprising an amino acid sequence depicted in SEQ ID NO:1 or 2, a
derivative thereof, or a partial peptide of the polypeptide or the
derivative.

Claims:

1. An antibody that recognizes a polypeptide comprising an amino acid
sequence represented by SEQ ID NO: 1 or 2, a derivative thereof or a
partial peptide thereof.

2. The antibody according to claim 1, which is a monoclonal antibody.

3. The antibody according to claim 1, which is a humanized antibody or a
human antibody.

4. The antibody according to claim 1, which is labeled.

5. The antibody according to claim 1, which has an activity of decreasing
the number of macrophages in white adipose tissue

6. The antibody according to claim 1, which has an activity of
ameliorating insulin resistance.

7. A hybridoma cell that produces the antibody according to claim 1.

8. A method for producing the antibody according to claim 1, comprising:
culturing a hybridoma cell that produces said antibody in vivo or in
vitro; and collecting the antibody according to claim 1 from the body
fluid or the culture.

9. A diagnostic agent comprising the antibody according to claim 1.

10. The diagnostic agent according to claim 9, which is used for
diagnosing type 2 diabetes and/or obesity.

11. A pharmaceutical agent comprising the antibody according to claim 1.

12. The pharmaceutical agent according to claim 11, which is an agent for
preventing and/or treating type 2 diabetes and/or obesity.

13. The pharmaceutical agent according to claim 11, which is an agent that
decreases the number of macrophages in white adipose tissue.

14. The pharmaceutical agent according to claim 11, which is an agent that
ameliorates insulin resistance.

15. A method for preventing and/or treating type 2 diabetes and/or
obesity, comprising administering an effective amount of the antibody
according to claim 1 to a mammal.

16. A method for decreasing the number of macrophages in white adipose
tissue, comprising administering an effective amount of the antibody
according to claim 1 to a mammal.

17. A method for ameliorating insulin resistance, comprising administering
an effective amount of the antibody according to claim 1 to a mammal.

18. A method for diagnosing type 2 diabetes and/or obesity which comprises
contacting an antibody according to claim 1 with a biological sample
obtained from a test subject

19. A method for producing an agent for preventing and/or treating type 2
diabetes and/or obesity which comprises incorporating an antibody
according to claim 1 into a formulation suitable for administration to a
patient

20. A method for producing an agent for decreasing the number of
macrophages in white adipose tissue which comprises incorporating an
antibody according to claim 1 into a formulation suitable for
administration to a patient

21. A method for producing an agent for ameliorating insulin resistance
which comprises incorporating an antibody according to claim 1 into a
formulation suitable for administration to a patient

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to an antibody that recognizes BRAK
(Breast and Kidney-expressed chemokine) and to use thereof.

BACKGROUND OF THE INVENTION

[0002]Recently, the number of diabetes patients whose onset is closely
related to obesity (particularly, "type 2 diabetes" that shows insulin
resistance) has been increasing worldwide. Accompanying this increase is
the prevailing trends in hypertension, arteriosclerosis and the like. The
pathogenic mechanisms for them presumably result from general
disturbances in the fat metabolism and hormonal regulation, which are now
collectively referred to as "metabolic syndrome".

[0003]According to recent reports, gene expression analyses using white
adipose tissue from obese mice and obese patients found that macrophages
in white adipose tissue play a key role in obesity and the accompanying
development of insulin resistance (see non-patent documents 1 and 2). In
experiments with obese mice, first, TNFα was produced from their
macrophages and suppressed glucose absorption by insulin. At the same
time, secretions of inflammatory cytokines such as IL-1 and IL-6
increased, leading to chronic inflammation (see non-patent documents 3
and 4). In fact, in mice deficient in TNFα or a receptor thereof,
insulin resistance caused by obesity was ameliorated (see non-patent
document 5).

[0004]Recent studies suggest that activations of kinase JNK and ER stress
signal transduction pathways are essential for insulin resistance caused
by obesity (see non-patent documents 6 and 7). In addition, blockage of
IKK-β/NF-κB pathway in the liver has also been reported to
ameliorate the insulin resistance caused by obesity (see non-patent
documents 8 and 9). Hence, activation of the ER stress and the JNK
pathway or the IKK-β/NF-κB pathway mentioned above caused by
increased secretion of inflammatory cytokine from obese adipose tissue or
load of fatty acid due to macrophage infiltration is assumed to be the
major cause of the development of insulin resistance associated with
obesity.

[0005]Furthermore, it is also reported that production of adiponectin, a
crucial secretory hormone for regulating lipid and glucose energy
metabolism, is suppressed in adipose tissue of obesity mice (see
non-patent document 10).

[0007]So far, more than 50 types of chemokine genes have been found
encoded on a mammalian chromosome. These chemokines consist of 92-99
amino acids (with a molecular weight of about 8,000-10,000), each
containing four cysteine residues that form disulfide bonds. The cysteine
residues and the amino acid sequence nearby the residues at the N-termini
are the characteristic features of the chemokines, based on which
chemokines are classified into four groups, namely "CC", "CXC",
"CX3C" and "C" (see non-patent document 15). Sixteen types of CXC
chemokines, in particular, have been identified so far and classified
into ELR.sup.+ CXC and ELR.sup.- CXC chemokines according to the presence
or the absence of Glu-Leu-Arg sequence (ELR sequence) at the N-termini.
BRAK belongs to ELR.sup.- CXC chemokines without the ELR sequence and is
universally referred to as "CXCL14".

[0008]Recent studies revealed that BRAK has an activity of allowing
specific migration of human peripheral blood monocyte-derived macrophages
activated by prostaglandin E2 (see non-patent document 16). BRAK was
also reported to allow specific migration of dendritic precursor cells
whose differentiation was induced by human monocytes and hematopoietic
precursor cells (see non-patent documents 17 and 18) and human breast
cancer-derived epithelial cell lines (see non-patent document 19). So
BRAK is a unique chemokine that induces migration of tissue macrophages,
dendritic precursor cells or the like rather than T cells, B cells or the
like (see non-patent document 16) and no other CXC chemokine is known to
have the same action spectrum.

[0009]Expression of BRAK has been confirmed in the small intestine,
kidney, liver, uterus, mammary gland and the like of human (see
non-patent document 14). Interestingly, BRAK expression was abrogated in
uterus or breast cancer patients' specimens and cancer cell lines (see
non-patent documents 11 and 12). This finding infers that BRAK-responsive
cells are less likely to migrate to cancer tissue (see non-patent
document 20). BRAK expression in adult mice is confirmed predominantly in
the brain, lung, skeletal muscle and ovary (see non-patent document 14).
There has been, however, no insight into behaviors and functions of
BRAK-responsive cells in mouse individuals, in particular, behaviors and
functions of monocytes and macrophages in white adipose tissue.

[0010]Accordingly, in order to reveal the relationship between chemotaxis
of the monocytes and the macrophages to the white adipose tissue and BARK
as well as to analyze dynamics of various parameters relating to obesity
and insulin response, we prepared knockout non-human animals having the
BRAK gene deleted, specifically BRAK knockout mice (BRAK-homo-deficient
mice (-/-)). With this BRAK knockout mice and control mice from the same
litter (wild-type mice (+/+), BRAK-hetero-deficient mice (+/-)), insulin
resistance and the number of macrophages in the white adipose tissue were
analyzed and compared (International Application No: PCT/JP2006/324622).

[0011]As a result, when BRAK knockout mice were fed on general diet, their
weights and the amounts of white fat were significantly less than those
of the control mice. When the mice were fed on high-fat diet, liver
enlargement and fatty livers characteristic of obese mice were alleviated
even though the amounts of white fat were similar to those of the control
mice. In addition, the numbers of macrophages in the white adipose tissue
of the BRAK knockout mice were significantly less compared to those of
the control mice, and no insulin resistance associated with obesity was
observed. Accordingly, we found that BRAK (CXCL14) was involved in
process of obesity process due to food intake regulation, migration of
macrophages to white adipose tissue, and acquirement of obese insulin
resistance, a typical symptom of type 2 diabetes.

[0033]Thus, the objectives of the present invention are to provide a
compound that suppresses or inhibits BRAK (CXCL14) functions (e.g.,
production or action of BRAK) and use thereof, more particularly, to
provide an antibody that recognizes BRAK and use thereof. Specifically,
the objective of the invention is to provide effective use of a compound
that suppresses or inhibits the BRAK functions (more particularly, an
antibody that specifically recognizes BRAK) for diagnosing the risk of
type 2 diabetes and for preventing and/or treating (alleviating) type 2
diabetes and obesity.

[0034]In order to solve the above problems, we have gone through keen
examination and consequently developed a compound (more particularly, an
antibody) capable of solving the problems above and use thereof (e.g.,
diagnosis, prevention and treatment of type 2 diabetes and obesity),
thereby accomplishing the present invention.

[0035]Thus, the present invention relates to the followings.

[0036](1) An antibody that recognizes a polypeptide comprising an amino
acid sequence represented by SEQ ID NO: 1 or 2, a derivative thereof, or
a partial peptide thereof.

[0037]Examples of the antibodies of the invention include monoclonal
antibodies, humanized or human antibodies, labeled antibodies and
combinations of these antibodies.

[0038]Examples of the antibodies of the invention also include antibodies
that have activities of decreasing the number of macrophages in white
adipose tissue and/or ameliorating insulin resistance.

[0039](2) A hybridoma cell that produces the antibody according to (1)
above.

[0040](3) A method for producing the antibody according to (1) above,
comprising: culturing the hybridoma cell according to (2) above in vivo
or in vitro; and collecting the antibody according to (1) above from the
body fluid or culture.

[0041](4) A diagnostic agent comprising the antibody according to (1)
above.

[0042]The diagnostic agent of the invention may be used for diagnosing,
for example, type 2 diabetes and/or obesity.

[0043](5) A pharmaceutical agent comprising the antibody according to (1)
above.

[0044]The pharmaceutical agent of the invention may be used, for example,
as an agent for preventing and/or treating type 2 diabetes and/or
obesity, as an agent that decreases the number of macrophages in white
adipose tissue, or as an agent that ameliorates insulin resistance.

[0045](6) A method for preventing and/or treating type 2 diabetes and/or
obesity, comprising administering an effective amount of the antibody
according to (1) above to a mammal.

[0046](7) A method for decreasing the number of macrophages in white
adipose tissue, comprising administering an effective amount of the
antibody according to (1) above to a mammal.

[0047](8) A method for ameliorating insulin resistance, comprising
administering an effective amount of the antibody according to (1) above
to a mammal.

[0048](9) Use of the antibody according to (1) above for producing an
agent for diagnosing type 2 diabetes and/or obesity. In addition, an
antibody according to (1) above for diagnosing type 2 diabetes and/or
obesity.

[0049](10) Use of the antibody according to (1) above for producing an
agent for preventing and/or treating type 2 diabetes and/or obesity. In
addition, an antibody according to (1) above for preventing and/or
treating type 2 diabetes and/or obesity.

[0050](11) Use of the antibody according to (1) above for producing an
agent for decreasing the number of macrophages in white adipose tissue.
In addition, an antibody according to (1) above for decreasing the number
of macrophages in white adipose tissue.

[0051](12) Use of the antibody according to (1) above for producing an
agent for ameliorating insulin resistance. In addition, an antibody
according to (1) above for ameliorating insulin resistance.

BRIEF DESCRIPTION OF THE DRAWING

[0052]FIG. 1 is data examining chemotaxis of C2C12 cells and
forskolin-stimulated C2C12 cells to CXCL14. After six hours of culture,
the number of cells that migrated from inside to the back of the
Chemotaxicell microchamber was determined to test statistical significant
difference. The graph shows average value±standard error for each
group.

[0053]FIG. 2 is data examining whether chemotaxis of forskolin-stimulated
C2C12 cells to CXCL14 is neutralized by addition of an anti-mouse
CXCL14-specific antibody. After six hours of culture, the number of cells
that migrated from inside to the back of the microchamber was determined
to test statistical significant difference. The graph shows average
value±standard error for each group.

[0054]FIG. 3 is data examining whether chemotaxis of forskolin-stimulated
THP-1 cells to CXCL14 is neutralized by addition of an anti-mouse
CXCL14-specific antibody. After two hours of culture, the number of cells
that migrated from inside to the back of the Chemotaxicell microchamber
was determined to test statistical significant difference. The graph
shows average value±standard error for each group.

[0055]FIG. 4 is data examining whether chemotaxis of forskolin-stimulated
THP-1 cells to CXCL14 is affected by addition of an anti-mouse CXCL14
monoclonal antibody. Typical pictures of stained cells that migrated from
inside to the back of the Chemotaxicell microchamber are shown.

[0056]FIG. 5 shows that an anti-mouse CXCL14-specific antibody neutralizes
an activity of CXCL14 inhibiting insulin signaling.
Differentiation-induced C2C12-derived myocytes were stimulated with
insulin in the presence of indicated substances and phosphorylation of
Akt Ser473 was determined by western blotting.

BEST MODES FOR CARRYING OUT THE INVENTION

[0057]Hereinafter, the present invention will be described in more detail.
The scope of the invention should not be limited to the description and
the present invention may appropriately be varied and carried out without
departing from the scope of the invention.

[0058]The present specification incorporates the entire content of the
specification of Japanese Patent Application Publication No. 2007-030027
based on which the present application claims priority. All of the
documents cited herein, for example, prior art documents, laid-open
patent publications, patent publications and other patent documents are
incorporated herein by reference.

[0059]Herein, BRAK and CXCL14 are synonymous and both terms are used in
the same sense.

[0060]1. Summary of the Invention

[0061]<Process of Achieving the Invention>

[0062]We noticed the increase of mRNA expression in the skeletal myocytes
of muscular dystrophy model mice mdx and focused on physiological
functions of BRAK (CXCL14).

[0063]Recently, macrophages have been reported successively to play an
important role in the repair of skeletal muscle injury, liver injury or
the like. Thus, we produced CXCL14(-/-) mdx and CXCL14(+/-) mdx mice by
mating CXCL14 gene-deficient mice [CXCL14(-/-) mice] with mdx mice.
Blood-Creatine kinase (CK) levels of these mice were determined
regularly. Although fluctuation was present among the individuals, the CK
levels of the CXCL14(-/-) mdx mice group were significantly lower than
those of the CXCL14(+/-) mdx mice group. However, when the skeletal
muscle and the diaphragm of aged CXCL14(-/-) mdx mice were
histochemically analyzed, myodegeneration unique to mdx mice occurred
equally likely to the aged CXCL14(+/-) mdx mice. In experiments of
skeletal muscle injury with habu snake venom injection, CXCL14(-/-) mdx
mice showed nearly equal kinetics of muscular tissue regeneration to that
of the normal mice. Based on these facts, decrease of blood-CK levels of
the CXCL14(-/-) mdx mice was seemingly not caused by alleviation of
fascial degeneration but it rather seemed to reflect different physiology
on the muscles of CXCL14.

[0064]Thus, the basic nature of the CXCL14(-/-) mdx mice was carefully
compared, the average weight of six-month-old CXCL14(-/-) female mice fed
on general diet was less than that of CXCL14(+/-) female mice by 25% and
the amount of white fat around uterus decreased to one-fourth. This
weight difference was caused by less food intake by the CXCL14(-/-) mice.
Next, high-fat diet was given to these mice. The CXCL14(-/-) mice
gradually become obese although they still weighed lighter than the
CXCL14(+/-) mice and the amounts of visceral white fat became nearly
equal. Liver enlargement and fatty liver unique to obese mice were
alleviated in these CXCL14(-/-) mice. Sugar levels of the CXCL14(-/-)
mice fed on high-fat diet were significantly lower than those of the
high-fat diet-fed CXCL14(+/-) mice. When the changes in sugar levels
following insulin administration were measured, insulin resistance of the
obese individuals was ameliorated for high-fat diet-fed CXCL14(-/-) mice.
From these results, we decided to analyze in more detail about the
physiological roles of CXCL14 in type 2 diabetes and obesity. Since the
amelioration of insulin resistance of CXCL14(-/-) mice was particularly
predominant in the skeletal muscle, the decrease in the CK values
observed in the CXCL14(-/-) mdx mice appeared to be closely related to
glucose metabolism.

[0065]<Mechanism of Actions of the Present Invention and BRAK>

[0066]Based on the results from the analysis using BRAK (CXCL14)
gene-deficient mouse strain [CXCL14(-/-) mice], we found out that CXCL14
was involved in the acquirement of obese insulin resistance, i.e., a
typical symptom of type 2 diabetes, and the obesity process due to food
intake regulation (these are described in more detail in (1) and (2)
below). From these findings, we found that a compound that suppresses or
inhibits the functions of CXCL14 (e.g., production or action of CXCL14),
in particular an antibody that specifically recognizes CXCL14, was
effective as a novel agent for treating type 2 diabetes and potentially
as an anti-obesity agent. We further found that the above-mentioned
compound (specifically, an anti-BRAK antibody) was also effective as a
diagnostic agent for type 2 diabetes and obesity risk.

[0067](1) Role of CXCL14 in Acquiring Obese Insulin Resistance

[0068]CXCL14 is a protein with 77 amino acids (99 amino acids if a signal
peptide is included) obtained by cloning chemokine cDNA that induces a
tissue macrophage (Hromas, R. et al., BBRC, 255: 703-706, 1999;
Frederick, M. J. et al., Am. J. Pathol., 156: 1937-1950, 2000; Sleeman,
M. A. et al., Int. Immunol., 12: 677-689, 2000). We found that
CXCL14(-/-) mice were less apt to become obese insulin resistant on
high-fat diet. Based on this fact, we assumed that the above-described
gene-modified mice (CXCL14(-/-) mice) were useful for research and
development of a therapeutic agent for type 2 diabetes (International
Application No: PCT/JP2006/324622).

[0069]CXCL14(+/-) mice were raised on high-fat diet for 12 weeks as
control mice. As with general overeating obese mice, 1) macrophage
infiltration into visceral white fat; 2) decrease in the blood
concentration of adiponectin; 3) increase in the blood concentration of
retinol binding protein 4 (RBP4), one of the causative factors of insulin
resistance; 4) fatty liver; and 5) slowing in decrease in the blood-sugar
levels upon abdominal administration of insulin, were observed in these
mice. Meanwhile, 1) to 5) above were all significantly improved in
CXCL14(-/-) mice fed on high-fat diet. This difference was particularly
prominent in female mice. Double genetic mutant mice were produced by
mating a transgenic mouse forcibly expressing CXCL14 in a skeletal
muscle-specific manner with a CXCL14(-/-) mouse. Insulin resistance due
to high-fat diet recovered in these double genetic mutant mice. On the
other hand, CXCL14 partially inhibited Akt kinase phosphorylation and
2-deoxy-glucose (analogue of glucose) intake caused by insulin
stimulation in an in vitro experimental system of insulin-stimulated
myotubes prepared by inducing differentiation of C2C12 myoblast lines.
These experimental results showed, for the first time, that CXCL14 not
only acts as a macrophage chemotactic factor but also directly regulates
sugar metabolism. Since insulin response was particularly improved in the
skeleton muscle of the CXCL14(-/-) mice fed on high-fat diet, CXCL14
appears to not only induce chronic inflammatory reaction due to
macrophage infiltration into visceral fat but also directly inhibit sugar
absorption by the skeleton muscle, thereby contributing to development of
obese insulin resistance.

[0070](2) Relationship Between CXCL14 and Obesity

[0071]The average weight of CXCL14(-/-) female mice was lighter than that
of CXCL14(+/-) female mice by 20-25% in general diet conditions as well
as high-fat diet conditions. The same was observed in double-mutant mice
of genetically-obese leptin-deficient mice ob/ob and CXCL14(-/-) mice.
While 8-10-week-old CXCL14(-/-) ob/ob female mice presented obvious
obesity, their weights lightened by 15-20% as compared to CXCL14(-/-)
ob/ob female mice and their food intake also decreased by approximately
20%. Thus, CXCL14 seemed to have a significant effect of increasing
appetite or suppressing loss of appetite by a mechanism independent from
leptin system.

[0072]2. Compound That Suppresses or Inhibits CXCL14 Functions

[0073]A compound that suppresses or inhibits CXCL14 functions may be, for
example, a compound that suppresses or inhibits production or action of
CXCL14. Examples of such compounds include (i) a compound that suppresses
or inhibits the functions of the CXCL14 gene, (ii) a compound that
suppresses or inhibits the CXCL14 functions by directly interacting
(e.g., binding or assembling) with a CXCL14 molecule, and (iii) a
compound that suppresses or inhibits the CXCL14 functions by interacting
(e.g., binding or assembling) with a substance (a substrate of CXCL14) or
a cell targeted by CXCL14. These compounds are not particularly limited
and may be either a so-called low- or high-molecular compound or a
compound having a molecular weight between them.

[0074]Specifically, examples of the compounds described in (i) above
include nucleic acids and other various organic compounds that suppress
or inhibit the functions of the CXCL14 gene. Such nucleic acids may be
any one of polynucleotides, oligonucleotides (DNA, RNA) and peptide
nucleic acids (PNA), specific types being, for example, antisense DNA,
siRNA, shRNA and microRNA.

[0075]Examples of the compounds described in (ii) above include antibodies
and enzymes that specifically recognize CXCL14, natural or synthetic
polypeptides and oligopeptides not included in such antibodies or
enzymes, and other various organic compounds.

[0076]The compounds of (iii) above may be referred to as compounds that
indirectly suppress or inhibit CXCL14 since they do not act directly on
CXCL14 and a gene thereof. Examples of such compounds include antibodies
and enzymes that specifically recognize a substance targeted by CXCL14,
natural or synthetic polypeptides and oligopeptides not included in such
antibodies or enzymes, and other various organic compounds.

[0077]The compounds described above that suppress or inhibit the CXCL14
functions may preferably be used as a diagnostic agent for type 2
diabetes and obesity, an agent or a method for preventing and/or treating
type 2 diabetes and obesity, an agent or a method for decreasing the
number of macrophages in the white adipose tissue and an agent or a
method for ameliorating insulin resistance.

[0078]Hereinafter, an "antibody that recognizes CXCL14", one of the
particularly favorable embodiments among the various compounds mentioned
above that suppress or inhibit the CXCL14 functions, will be described in
detail including its use in various applications. Similar description may
apply to other compounds as to the use in various applications.

[0079]3. Antibody of the Invention

[0080]SEQ ID NOS: 1-6 among the sequences identified herein represent the
amino acid sequences of the following peptides. The amino acid sequences
(99 amino acids) represented by SEQ ID NOS: 1 and 2 each have a signal
sequence of 22 amino acids at the N terminus. The amino acid sequences
represented by SEQ ID NOS: 1 and 2 have been assigned "Accession number:
AAD03839" and "Accession number: AAD34157" on the website of NCBI
(GenBank) (http://www.ncbi.nlm.nih.gov/), respectively. The amino acid
sequences represented by SEQ ID NOS: 3-6 are each a part of the amino
acid sequences represented by SEQ ID NOS: 1 and 2 while the numerical
range in the parentheses indicates their positions (indicated as the
number of amino acid residues counted from the N terminus) in the amino
acid sequence represented by SEQ ID NOS: 1 and 2.

[0081][SEQ ID NO: 1] human CXCL14

[0082][SEQ ID NO: 2] mouse CXCL14

[0083][SEQ ID NO: 3] human CXCL14 (23-99)

[0084][SEQ ID NO: 4] human CXCL14 (24-35)

[0085][SEQ ID NO: 5] mouse CXCL14 (23-99)

[0086][SEQ ID NO: 6] mouse CXCL14 (24-35)

[0087]Below are the human CXCL14 amino acid sequence represented by SEQ ID
NO: 1 and the mouse CXCL14 amino acid sequence represented by SEQ ID NO:
2 (both given in single letter codes for the amino acids). In these amino
acid sequences, the underlined amino acid residues (total of two
residues) are the difference between the human CXCL14 and the mouse
CXCL14 in parts other than the signal sequence.

[0088]Proteins (polypeptides or partial peptides) mentioned in this
specification have the N terminus (amino terminus) on the left side and
the C terminus (carboxyl terminus) on the right side in accordance with
the custom of peptide representation. The proteins used with the
invention such as polypeptides comprising the amino acid sequence
represented by SEQ ID NO: 1 may have any of a carboxyl group,
carboxylate, amide or ester at the C terminus.

[0089]Examples of derivatives of polypeptides recognizable by the antibody
of the invention, namely human or mouse CXCL14 derivatives, include those
in which the amino acid residues of the amino acid sequence represented
by SEQ ID NO: 1 or 2 are partially substituted with a replaceable
group(s), partially deleted, or partially added or inserted with an amino
acid residue(s). Examples of derivatives of polypeptides having the amino
acid sequence represented by SEQ ID NO: 1 or 2 include those having one
or more amino acids (preferably, approximately 1-10, more preferably
several (1-5) and still more preferably 1, 2 or 3 amino acids) deleted,
those having one or more amino acids (preferably, approximately 1-20
amino acids, more preferably approximately 1-10, more preferably several
(1-5) and still more preferably 1, 2 or 3 amino acids) added to the amino
acid sequence above, those having one or more amino acids (preferably,
approximately 1-20, more preferably approximately 1-10, more preferably
several (1-5) and still more preferably 1, 2 or 3 amino acids) inserted
into the amino acid sequence above, or those having one or more amino
acids (preferably, approximately 1-10, more preferably several (1-5) and
still more preferably 1, 2 or 3 amino acids) substituted with other amino
acids.

[0091]Examples of partial peptides recognizable by the antibody of the
invention, namely partial peptides of human or mouse CXCL14 or
derivatives thereof, include polypeptides having the amino acid residues
partially deleted, partially substituted with a replaceable group(s)
(e.g., Cys, hydroxyl group, etc.), or partially deleted and partially
substituted with a replaceable group(s) (e.g., Cys, hydroxyl group, etc.)
in the amino acid sequence represented by SEQ ID NO: 1 or 2.

[0092]Examples of such partial peptides include those having approximately
22 residues (signal peptide portion) at the N-terminus of the human or
mouse CXCL14 or derivatives thereof deleted. More specifically, such
partial peptides are preferably (i) a polypeptide having the amino acids
23-99 (SEQ ID NO: 3) of the amino acid sequence represented by SEQ ID NO:
1; (ii) a polypeptide having the amino acids 24-35 (SEQ ID NO: 4) of the
amino acid sequence represented by SEQ ID NO: 1; (iii) those having a
part (e.g., one) of the amino acid residues of these polypeptides of (i)
and (ii) substituted with replaceable group(s); (iv) a polypeptide having
the amino acids 23-99 (SEQ ID NO: 5) of the amino acid sequence
represented by SEQ ID NO: 2; (v) a polypeptide having the amino acids
24-35 (SEQ ID NO: 6) of the amino acid sequence represented by SEQ ID NO:
2; and (vi) those having a part (e.g., one) of the amino acid residues of
these polypeptides of (iv) and (v) substituted with a replaceable
group(s).

[0093]A method for preparing an antigen to an antibody and a method for
producing an antibody may be a known method such as a method described in
WO 94/17197 or a method based thereon. Hereinafter, an exemplary method
will be described.

[0094](1) Preparation of Antigen

[0095]An antigen used for preparing an antibody of the invention may be,
for example, CXCL14 (BRAK), a derivative thereof, a partial peptide
thereof, or a synthetic peptide having one or more types of antigen
determinants identical to those of CXCL14 (hereinafter, these are simply
referred to as "CXCL14 antigens"). CXCL14, a derivative thereof or a
partial peptide thereof may be those described above. A CXCL14 antigen
may be prepared from a mammal such as human, mouse, possibly monkey, rat
or swine by a known method or a method based thereon. Alternatively, a
CXCL14 antigen used may be a commercially available natural purified
preparation or a synthetic peptide.

[0096]A CXCL14 antigen may be prepared by a known method such as a method
described in WO02/06483. A CXCL14 antigen may also be prepared (a) by a
known method or a method based thereon from tissue or a cell from a
mammal such as human, mouse, possibly monkey, rat or swine; (b) by
chemical synthesis according to a known peptide synthesis method with a
peptide synthesizer or the like; or (c) by culturing a transformant
containing DNA coding for CXCL14 or a derivative thereof.

[0097](a) Where a CXCL14 antigen is prepared from mammal tissue or cell,
the tissue or the cell is homogenized, extracted with an acid or an
alcohol, and the extract is purified and isolated by a combination of
chromatographies such as salting-out, dialysis, gel filtration,
reversed-phase chromatography, ion-exchange chromatography or affinity
chromatography, thereby preparing a CXCL14 antigen.

[0098](b) A synthetic peptide used for chemically synthesizing a CXCL14
antigen may be, for example, one having the same structure as a native
purified CXCL14 antigen, or a peptide containing one or more types of
amino acid sequences identical to amino acid sequences having 3 or more,
preferably 6 or more amino acids of the amino acid sequence of CXCL14.

[0099](c) Where a CXCL14 antigen is produced with a transformant
containing DNA, the DNA may be prepared according to a known cloning
method (e.g., a method described in Molecular Cloning (2nd ed.; J.
Sambrook et al., Cold Spring Harbor Lab. Press, 1989)). Examples of such
cloning method include: (1) a method for obtaining a transformant
containing DNA coding for a CXCL14 antigen from a cDNA library using a
DNA probe or DNA primers designed based on the amino acid sequence of the
CXCL14 antigen by hybridization; or (2) a method for obtaining a
transformant containing DNA coding for a CXCL14 antigen by PCR using DNA
primers designed based on an amino acid sequence of the CXCL14 antigen.

[0100]A partial peptide obtained by CXCL14 hydrolysis may be, for example,
a partial peptide or a mixture thereof obtained by sequentially
hydrolyzing a polypeptide having the amino acid sequence represented by
SEQ ID NO: 1 or 2 from the N- and/or the C-terminus with exoprotease such
as aminopeptidase or carboxypeptidase, or a partial peptide or a mixture
thereof obtained by hydrolyzing a polypeptide having the amino acid
sequence represented by SEQ ID NO: 1 or 2 with various endopeptidases.

[0101]The synthetic peptide may be produced by a known routine procedure
which may be either solid- or liquid-phase synthesis. Specifically, a
partial peptide or amino acids that may constitute this peptide and the
remaining part are condensed and a protecting group of the product, if
any, is detached, thereby producing a peptide of interest. Such a
condensation method and a detachment method of the protecting group are,
for example, described in B. Merrifield [Journal of American Chemical
Society (J. Am. Chem. Soc.), 85, 2149, 1963], M. Bodanszky and M. A.
Ondetti [Peptide Synthesis, Interscience publishers, New York 1966],
Schroeder and Luebke [The Peptide, Academic Press, New York, 1965], Nobuo
Izumiya et al. [Fundamentals and Experiments of Peptide Synthesis,
Maruzen, 1985], and Haruaki Yajima and Shunpei Yanagihara [Seikagaku
Jikken Kouza I: Tanpakusitu no Kagaku IV (Course for Biochemical
Experiments 1: Chemistry of Protein IV), 205, 1977]. After the reaction,
the peptide may be purified by combining general purification methods
such as solvent extraction, distillation, column chromatography, liquid
chromatography, recrystallization or the like. If the peptide obtained by
the above method is a free body, it may be transformed into an
appropriate salt by a known method whereas if the peptide is obtained as
a salt, it may be transformed into a free body by a known method.

[0102]An amide body of the peptide may be obtained by using a commercially
available resin for peptide synthesis suitable for amide formation.
Examples of such resins include a chloromethyl resin, a hydroxymethyl
resin, a benzhydrylamine resin, an aminomethyl resin, a 4-benzyloxybenzyl
alcohol resin, a 4-methylbenzhydrylamine resin, a PAM resin, a
4-hydroxymethyl methylphenyl acetamidemethyl resin, a polyacrylamide
resin, a 4-(2',4'-dimetoxylphenyl-hydroxymethyl)phenoxy resin, and a
4-(2',4'-dimetoxylphenyl-Fmoc aminoethyl)phenoxy resin. With the use of
such a resin, amino acids having an alpha-amino group and a side-chain
functional group appropriately protected are condensed on the resin
following the sequence of the peptide of interest according to one of
various condensation methods. At the end of the reaction, each protecting
group is removed upon peptide cleavage from the resin. Alternatively, a
peptide of interest may also be obtained by taking out a part of the
protected peptide with a chlorotrityl resin, an oxime resin,
4-hydroxybenzoic acid resin or the like, and further removing the
protecting group by a routine procedure.

[0103]Various activating reagents available for peptide synthesis may be
used for the condensation of the protected amino acids, although
carbodiimides are preferably used. Examples of such carbodiimides include
DCC, N,N'-diisopropylcarbodiimide and
N-ethyl-N'-(3-dimethylaminoprolyl)carbodiimide. For activation with each
activating reagent, the protected amino acid may be directly added to the
resin with a racemization suppressant (e.g., HOBt, HOOBt, etc.) or an
activated amino acid that has been protected beforehand as a symmetric
anhydride, HOBt ester or HOOBt ester may be added to the resin. A solvent
used for the activation of the protected amino acid and the condensation
with the resin may be appropriately selected from known solvents
available for peptide condensation reaction. Examples of such solvents
used for activation of protected amino acids or condensation with a resin
include acid amides such as N,N-dimethylformamide, N,N-dimethylacetamide
and N-methylpyrrolidone, halogenated hydrocarbons such as methylene
chloride and chloroform, alcohols such as trifluoroethanol, sulfoxides
such as dimethylsulfoxide, tertiary amines such as pyridine, ethers such
as dioxane and tetrahydrofuran, nitriles such as acetonitrile and
propionitrile, esters such as methyl acetate and ethyl acetate, and an
appropriate mixture thereof. A reaction temperature is appropriately
selected from a range known for peptide binding reaction, generally
selected from a range of about -20° C. to about 50° C. The
activated amino acid derivatives are usually used in an about 1.5- to
about 4-fold excess. If the condensation is inadequate when tested by
ninhydrin reaction, the condensation reaction may be repeated without
removing the protecting groups so as to achieve adequate condensation.
When the condensation is inadequate even after repetitive reactions, the
unreacted amino acids are acetylated with acetic anhydride or
acetylimidazole so as not to affect the subsequent reactions.

[0104]Examples of the protecting groups for amino groups of the raw amino
acids include a Z group, a Boc group, a tertiary pentyloxycarbonyl group,
an isobornyloxycarbonyl group, a 4-methoxybenzyloxycarbonyl group, a Cl-Z
group, a Br-Z group, an adamantyloxycarbonyl group, a trifluoroacetyl
group, a phthaloyl group, a formyl group, a 2-nitrophenylsulfenyl group,
a diphenylphosphinothioyl group and a Fmoc group. Examples of protecting
groups of carboxy groups include a C1-6 alkyl group, a C3-8
cycloalkyl group and C7-14 aralkyl group as well as a 2-adamantyl
group, a 4-nitrobenzyl group, a 4-methoxybenzyl group, a 4-chlorobenzyl
group, a phenacyl group and a benzyloxycarbonyl hydrazide group, a
tertiary butoxycarbonyl hydrazide group and a trityl hydrazide group.

[0105]A hydroxyl group for serine or threonine may be protected, for
example, by esterification or etherification. Examples of groups suitable
for this esterification include lower (C1-6) alkanoyl groups such as
an acetyl group, aroyl groups such as a benzoyl group and carbon-derived
groups such as a benzyloxycarbonyl group and an ethoxycarbonyl group.
Groups suitable for etherification are, for example, a benzyl group, a
tetrahydropyranyl group and a tertiary butyl group.

[0106]Examples of protecting groups for a phenolic hydroxyl group of
tyrosine include a Bzl group, a Cl-Bzl group, a 2-nitrobenzyl group, a
Br-Z group and a tertiary butyl group.

[0107]Examples of protecting groups for imidazole groups of histidine
include a Tos group, a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, a
DNP group, a Born group, a Bum group, a Boc group, a Trt group and a Fmoc
group.

[0108]Examples of a raw material with an activated carboxyl group include
corresponding acid anhydride, azide and active esters [esters with
alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol,
2,4-dinitrophenol, cyanomethyl alcohol, para-nitrophenol, HONB,
N-hydroxysuccinimide, N-hydroxyphthalimide and HOBt)]. An example of a
raw material with an activated amino group includes corresponding amide
phosphate.

[0109]Examples of a method for removing (detaching) a protecting group
include a catalytic reduction method using a hydrogen stream in the
presence of a catalyst such as Pd-black or Pd-carbon, an acid treatment
with anhydrous hydrogen fluoride, methanesulfonic acid,
trifluoromethanesulfonic acid, trifluoroacetic acid, a mixture thereof or
the like, a base treatment with diisopropylethylamine, triethylamine,
piperidine, piperazine or the like, and reduction with sodium in liquid
ammonia. Detachment reaction by an acid treatment mentioned above is
generally carried out at a temperature of -20° C. to 40° C.
while addition of a cation capture agent such as anisole, phenol,
thioanisole, meta-cresol, para-cresol, dimethyl sulfide,
1,4-butanedithiol or 1,2-ethanedithiol is effective. Moreover, a
2,4-dinitrophenyl group used as an imidazole-protecting group of
histidine is removed by a thiophenol treatment while a formyl group used
as an indole-protecting group for tryptophan may be removed not only by
deprotection by an acid treatment in the presence of 1,2-ethanedithiol,
1,4-butanedithiole or the like mentioned above but also by an alkali
treatment with diluted sodium hydrate, diluted ammonia or the like.

[0110]Protection and protecting groups for functional groups that should
not be involved with the reaction of the raw material, detachment of
these protecting groups, and activation of functional groups involved in
the reaction may be appropriately selected from known groups or known
procedures.

[0111]Another method for obtaining an amide body of a peptide includes:
first, amidating the alpha-carboxyl group of the carboxy-terminal amino
acid; then extending a peptide chain to an intended length from the amino
group side; producing a peptide having the protecting group removed only
for the alpha-amino group at the N-terminus of the peptide chain as well
as a peptide (or an amino acid) having the protecting group removed only
for the carboxyl group at the C-terminus; and condensing these peptides
in a mixed solvent as described above. Details on condensation reaction
are the same as described above. After purifying the protected peptide
resulting from condensation, all of the protecting groups are removed by
the method described above, thereby obtaining crude peptide of interest.
This crude peptide may be purified by any of the known various
purification procedures and the main fraction may be freeze-dried,
thereby obtaining an amide body of the peptide of interest.

[0112]In order to obtain an ester body of a peptide, an alpha-carboxyl
group of a carboxy-terminal amino acid is condensed with a suitable
alcohol to give an amino acid ester, with which an ester body of the
peptide of interest can be obtained in the same manner as the amide body
of the peptide.

[0113]A CXCL14 antigen that has been insolubilized may be immunized
directly. Alternatively, a complex of a CXCL14 antigen binding to or
adsorbed onto an appropriate carrier may be immunized. A mix ratio of the
carrier and the CXCL14 antigen (hapten) may be any ratio as long as an
antibody is efficiently produced against the CXCL14 antigen bound to or
adsorbed onto the carrier. Usually, a polymeric carrier commonly used for
producing an antibody to the hapten is used in 0.1-100 parts to a part of
hapten. Such a polymeric carrier may be, for example, a natural polymeric
carrier or a synthetic polymeric carrier. Examples of natural polymeric
carriers include serum albumin derived from a mammal such as bovine,
rabbit, human or the like, thyroglobulin from a mammal such as bovine,
rabbit or the like, hemoglobin from a mammal such as bovine, rabbit,
human, sheep or the like, and KHL (keyhole limpet) hemocyanin. Examples
of synthetic polymeric carriers include various latexes like polymers or
copolymers such as polyamino acids, polystyrenes, polyacryls, polyvinyls
and polypropylenes.

[0114]Various condensation agents may be used for coupling the hapten and
the carrier. Examples of conveniently used condensation agents include
diazonium compounds such as bis-diazotized benzidine that cross-links
tyrosine, histidine and tryptophan, dialdehyde compounds such as
glutaraldehyde that cross-links amino groups with each other,
diisocyanate compounds such as toluene-2,4-diisocyanate, dimaleimide
compounds such as N,N'-o-phenylenedimaleimide that cross-links thiol
groups with each other, a maleimide active ester compound that
cross-links an amino group and a thiol group, and a carbodiimide
compounds that cross-links an amino group and a carboxyl group.
Alternatively, in order to cross-link amino groups with each other, an
active ester reagent (e.g., SPDP, etc.) having a dithiopyridyl group is
reacted with one amino group and subsequently reduced to introduce a
thiol group while a maleimide group is introduced into the other amino
group with a maleimide active ester reagent, and then both amino groups
are reacted.

[0115](2) Preparation of Monoclonal Antibody

[0116]Although an antibody of the invention is not particularly limited
and it may be either a polyclonal antibody or a monoclonal antibody, it
is preferably a monoclonal antibody. Hereinafter, a method for preparing
an antibody of the invention will be described taking the case of a
monoclonal antibody as an example.

[0117]A CXCL14 antigen is administered to a warm-blooded animal, for
example, through intraperitoneal injection, intravenous injection,
subcutaneous injection or the like, alone or together with a carrier and
a diluent, to a site that allows antibody production. In order to enhance
the antibody production upon administration, a complete Freund's adjuvant
or an incomplete Freund's adjuvant may be administered. Administration is
usually performed once in every 2-6 weeks for a total of about 2-10
times. Although examples of warm-blooded animals include monkey, rabbit,
dog, guinea pig, mouse, rat, sheep, goat and chicken, a mouse is
preferably used for a production of an antibody, especially a monoclonal
antibody.

[0118]For production of a monoclonal antibody, individuals with positive
antibody titers are selected from the CXCL14 antigen-immunized
warm-blooded animals such as mice. Two to five days following the final
immunization, spleens or lymph nodes are collected from these individuals
to fuse antibody-producing cells contained therein with myeloma cells,
thereby preparing anti-CXCL14 monoclonal antibody-producing hybridomas.
The anti-CXCL14 antibody titer in a serum may be measured, for example,
by allowing a labeled CXCL14 described below to react with an antiserum
and determining the activity of the labeling agent bound to the antibody.
The fusion procedure may be carried out by a known method such as Kohler
and Milstein [Nature, 256, 495 (1975)]. Examples of fusion accelerators
include polyethyleneglycol (PEG) and Sendai virus, while PEG is
preferably used. Examples of myeloma cells include NS-1, P3U1, SP2/0 and
AP-1, while P3U1 is preferably used. A favorable ratio of the number of
the antibody-producing cells (spleen cells) used to the number of bone
marrow cells is generally about 1:1 to 20:1 where PEG (preferably PEG1000
to PEG6000) is added at a concentration of about 10-80% and incubated
generally at 20-40° C., preferably 30-37° C., generally for
1-10 minutes for carrying out efficient cell fusion.

[0119]Various methods may be employed for screening an anti-CXCL14
antibody-producing hybridoma. In an exemplary method, a hybridoma culture
supernatant is added directly or with a carrier to CXCL14, a derivative
thereof or a partial peptide thereof adsorbed onto a solid phase (e.g., a
microplate) and then an anti-immunoglobulin antibody (an anti-mouse
immunoglobulin antibody is used if mouse cells are used for the cell
fusion) labeled with a radioactive substance or an enzyme or protein A is
added to detect anti-CXCL14 monoclonal antibodies bound to the solid
phase. In another exemplary method, a hybridoma culture supernatant and
then CXCL14 labeled with a radioactive substance or an enzyme is added to
an anti-immunoglobulin antibody or protein A adsorbed onto a solid phase
to detect CXCL14 monoclonal antibodies bound on the solid phase.
Screening and production of the anti-CXCL14 monoclonal antibodies are
usually performed by adding HAT (hypoxanthine, aminopterin and thymidine)
in a medium for animal cells (e.g., RPMI1640) containing 10-20% fetal
bovine serum. An antibody titer of a hybridoma culture supernatant may be
measured in the same manner as the above-described measurement for an
anti-CXCL14 antibody titer in an antiserum.

[0120]Similar to usual separation and purification of a polyclonal
antibody, separation and purification of an anti-CXCL14 monoclonal
antibody is carried out by following a method for separating and
purifying an immunoglobulin (e.g., salting-out method, alcohol
precipitation method, isoelectric precipitation method, electrophoresis,
adsorption-desorption on an ion exchanger (e.g., DEAE),
ultracentrifugation method, gel filtration method, or specific
purification in which the antibody is collected with an antigen-binding
solid phase or an active adsorbent such as protein A or protein G
followed by dissociation of the binding).

[0121]Furthermore, sorting between a hybridoma producing an anti-CXCL14
antibody that reacts with a partial region of CXCL14 and a hybridoma
producing an anti-CXCL14 monoclonal antibody that reacts with CXCL14 but
not with the said partial region may be carried out, for example, by
determining the binding capacity between the peptide corresponding to
that partial region and the antibody produced by the hybridoma.

[0122]Hence, an antibody of the invention may be produced by culturing a
hybridoma cell in vivo in a warm-blooded animal or in vitro and
collecting an antibody from its fluid or culture.

[0123]The resulting antibody of the invention may be used as an agent for
diagnosing type 2 diabetes and obesity, an agent for preventing and/or
treating type 2 diabetes and obesity, an agent that decreases the number
of macrophages in white adipose tissue, or an agent that ameliorates
insulin resistance.

[0124]For these purposes, the antibody of the invention may be used as an
antibody molecule itself or as a fragment of the antibody or a
single-chain antibody of the V region, and all of them are within the
scope of the antibody of the invention. A fragment of the antibody refers
to a partial region of the antibody, specific examples being
F(ab')2, Fab', Fab, Fv (variable fragment of antibody), sFv, dsFv
(disulphide stabilized Fv) and dAb (single region antibody). A
single-chain antibody of the V region comprises VL (light-chain
variable region) and VH (heavy-chain variable region) combined by a
linker.

[0125]A preferable embodiment of an antibody of the invention comprises a
humanized antibody or a human antibody. These humanized and human
antibodies may collectively be referred to herein as "human antibodies"
or as "human monoclonal antibodies" in the case of monoclonal antibodies.
Similar to general preparation of a monoclonal antibody, these antibodies
may be prepared by immunizing a mammal having its immune system replaced
with a human immune system.

[0126]In order to prepare a humanized antibody, a complementarity
determining region (CDR) is grafted from a variable region of an antibody
of a mammal (non-human animal) such as a mouse into a human variable
region to restructure a variable region in which a human-derived
framework region (FR) and a non-human animal-derived CDR are used. Then,
this restructured variable region is linked to a human constant region,
thereby preparing a humanized antibody. A humanized antibody may also be
prepared as a chimeric antibody comprising a variable region from a
non-human antibody and a constant region from a human antibody.
Preparation of a humanized antibody is well known in the art.

[0127]Generally, specificity and binding affinity of an antigen-binding
part of the V region, i.e., a hypervariable region, matter for a human
antibody, but structurally it may be prepared from any animal. On the
other hand, structures of other parts of the V region and the constant
region preferably have the same structure as those of a human antibody. A
genetic engineering method for preparing a genetic sequence common to
human has been established.

[0128]4. Applications of Antibody of the Invention

[0129]Hereinafter, applications of an antibody of the invention will be
described in detail.

[0130](1) A diagnostic agent comprising an antibody of the invention

[0131]An antibody (particularly, a human monoclonal antibody) of the
invention may be used as an agent for diagnosing a disease associated
with CXCL14 (BRAK), namely type 2 diabetes and/or obesity.

[0132]Specifically, an antibody of the invention may be used for the
purpose of diagnosing the risk of type 2 diabetes and/or obesity. As
described above, based on the results from the analysis with
CXCL14-homo-deficient mice, CXCL14 was found to be involved in an
acquirement of obese insulin resistance (a typical symptom of type 2
diabetes) and a process of obesity due to food intake regulation. Thus,
an antigen level (CXCL14 level) in a biological sample such as a fluid,
tissue or the like collected from a test subject (a measured solution or
a test solution) is measured to assess the risk of the test subject to
develop type 2 diabetes or obesity.

[0133]Measurement using the antibody of the invention is not particularly
limited. Any measurement method may be employed as long as the amount of
the antibody, the antigen or the antibody-antigen complex corresponding
to the antigen level (CXCL14 level) in the measured solution is detected
by chemical or physical means, and calculated with a standard solution
containing a known level of antigen. Examples of such measurement methods
include a sandwich assay, a competitive assay, an immunometric assay and
nefelometry, while a sandwich assay or a competitive assay described
below is preferable, particularly a sandwich assay, in terms of
sensitivity and specificity.

[0134](A) Sandwich Assay

[0135]A sandwich assay is a method for quantifying CXCL14 or a derivative
thereof in a test solution by allowing reaction of an antibody of the
invention insolubilized on a carrier (a solid-phased antibody), a labeled
antibody of the invention (antibody with different epitope from that of
the solid-phased antibody: labeled antibody) and a test solution, and
then determining the activity of the labeling agent.

[0136]According to a sandwich assay, a test solution is allowed to react
with an antibody of the invention insolubilized on a carrier (primary
reaction), followed by reaction with a labeled antibody of the invention
(secondary reaction). Subsequently, the activity of the labeling agent on
the insolubilized carrier is determined to quantify the CXCL14 level in
the test solution. The primary and secondary reactions may be conducted
simultaneously or at different times. The labeling agent and the
insolubilization method may be similar to those described previously.
Moreover, in an immunoassay employing a sandwich assay, an antibody for a
solid phase and an antibody for labeling are not necessarily limited to a
single type, and a mixture of two or more types of antibodies may be used
for the purpose of enhancing the measurement sensitivity or the like. For
CXCL14 measurement by a sandwich assay, for example, when the antibody
used for the primary reaction recognizes a partial peptide on the
C-terminus of CXCL14 or a derivative thereof, the antibody used for the
secondary reaction is preferably an antibody that recognizes a part other
than the said partial peptide at the C-terminus (i.e., N-terminus). When
the antibody used for the primary reaction recognizes a partial peptide
on the N-terminus of CXCL14 or a derivative thereof, the antibody used
for the secondary reaction is preferably an antibody that recognizes a
part other than the said partial peptide at the N-terminus (i.e.,
C-terminus). The labeled antibody is preferably labeled with horseradish
peroxidase (HRP).

[0137](B) Competitive Assay

[0138]A competitive assay is a method for quantifying CXCL14 or a
derivative thereof in a test solution by competitively reacting the
antibody of the invention, a test solution and a labeled CXCL14 or a
derivative thereof, and determining the proportion of the labeled CXCL14
or the derivative thereof bound to the antibody. Quantification of CXCL14
or a derivative thereof in the test solution by a competitive assay is
preferably carried out by using, for example, a solid phase technique. A
specific example of a solid phase technique includes a method comprising:
using an anti-mouse IgG antibody (from ICN/CAPPEL) as a solid-phased
antibody; adding (i) an antibody of the invention, (ii) a peptide
represented by SEQ ID NO: 1 or 2 labeled with HRP and (iii) a test
solution to the plate having the solid-phased antibody thereon; and,
following reaction, determining the activity of HRP adsorbed onto the
solid phase, thereby quantifying CXCL14 or a derivative thereof.

[0139](C) Immunometric Assay

[0140]According to an immunometric assay, an antigen in a test solution
and a solid-phased antigen are allowed to competitively react with a
certain amount of a labeled antibody of the invention, followed by
separation between the solid phase and the liquid phase. Alternatively,
an antigen in a test solution is allowed to react with an excess amount
of a labeled antibody of the invention, then a solid-phased antigen is
added to bind the unreacted labeled antibody of the invention to the
solid phase, followed by separation between the solid phase and the
liquid phase. Subsequently, the amount of the label in either of the
phases is determined to quantify the antigen level in the test solution.

[0141](D) Nefelometry

[0142]According to nefelometry, the amount of insoluble precipitation
resulting from antigen-antibody reaction in a gel or a solution is
measured. When the antigen level in the test solution is low and the
amount of precipitation is small, laser nefelometry that utilizes laser
scattering is preferably used.

[0143]In the measurement methods of (A) to (D) above, a labeling agent
used for the methods using a labeling substance is, but not limited to, a
radioisotope, an enzyme, a fluorescent substance, a luminescent substance
or the like. Preferable examples of radioisotopes include, but not
limited to, [125I], [131I], [3H] and [14C]. The
above-mentioned enzymes are preferably, but not limited to, those that
are stable and that have high specific activity, examples being
beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase
and malate dehydrogenase. Examples of the above-mentioned fluorescent
substance include, but not limited to, fluorescamine and fluorescein
isothiocyanate. Examples of the above-mentioned luminescent substance
include, but not limited to, luminol, luminol derivatives, luciferin and
lucigenin. For binding between the antigen and the labeling agent, a
biotin-avidin compound may be used.

[0144]For insolubilization of an antigen or an antibody, physical
adsorption may be employed, or alternatively a method employing chemical
adsorption generally used for insolubilization or immobilization of a
protein, an enzyme or the like may be used. Examples of carriers include
insoluble polysaccharides such as agarose, dextran and cellulose,
synthetic resins such as polystyrene, polyacrylamide and silicon, and
glass.

[0146]The antibody of the invention may also be used for preparing an
antibody column for purifying CXCL14 or a derivative thereof, for
detecting CXCL14 or a derivative thereof in each fraction upon
purification, for analyzing behavior of CXCL14 or a derivative thereof in
a test cell or the like.

[0147](2) Pharmaceutical Agent Comprising Antibody of the Invention

[0148]An antibody of the invention (particularly a human monoclonal
antibody) may be used as an active component of a pharmaceutical agent
such as a prophylactic/therapeutic agent for diseases associated with
CXCL14 (BRAK), i.e., type 2 diabetes and/or obesity. Preferably, examples
of such pharmaceutical agents further include an agent for decreasing
macrophages in white adipose tissue and an agent for ameliorating insulin
resistance.

[0149]Hereinafter, use of a pharmaceutical agent as such
prophylactic/therapeutic agent will be described. Similar description may
apply to use of such decreasing and ameliorating agents mentioned above.

[0150]The prophylactic/therapeutic agent comprising an antibody of the
invention have low toxicity and may be parenterally or orally
administered to a human or a mammal (e.g., mouse) directly as a liquid
agent or as a pharmaceutical composition in an appropriate formulation.
An antibody of the invention may be administered by itself or as an
appropriate pharmaceutical composition. A pharmaceutical composition used
for administration may comprise an antibody of the invention or a salt
thereof with a pharmacologically-acceptable carrier, diluent or
excipient. Such a pharmaceutical composition may be provided as a
formation suitable for oral or parenteral administration. A parenteral
composition may be, for example, an injection, a suppository or the or
like, where the injection may comprise a formulation such as an
intravenous injection, a subcutaneous injection, an intradermal
injection, an intramuscular injection, a drip injection or the like. Such
injections may be prepared according to a known method. According to an
exemplary method, an injection may be prepared by dissolving, suspending
or emulsifying the antibody of the invention or a salt thereof in a
sterile aqueous or oily solution generally used for an injection.
Examples of injectable aqueous solutions include isotonic solutions
containing physiological saline, glucose, or other adjuvants, which may
be used together with an appropriate solubilizing agent such as an
alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,
polyethylene glycol), a nonionic surfactant (e.g., Polysorbate 80, HCO-50
(polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) or the
like. Examples of oily solutions include a sesame oil, a soybean oil and
the like while benzyl benzoate, benzyl alcohol or the like may be used
together as a solubilizing agent. The prepared injection is preferably
packed into an appropriate ampoule. A suppository used for rectal
administration may be prepared by mixing the antibody above or a salt
thereof with a general suppository base.

[0151]Examples of compositions for oral administration include solid or
liquid formulations, specifically, tablets (including sugar-coated
tablets and film-coated tablets), pills, granule, powder, capsules
(including soft capsules), syrups, emulsions, suspensions and the like.
Such compositions may be produced according to a known method and may
contain a carrier, a diluent or an excipient generally used in the art. A
carrier or an excipient used for a tablet may be, for example, lactose,
starch, sucrose and magnesium stearate.

[0152]The parenteral or oral pharmaceutical compositions described above
are favorably prepared into unit formulations that comply with the dosage
of the active element. Examples of such dosage unit formations include
tablets, pills, capsules, injections (ampoules) and suppositories.
Preferably, the content of the antibody is usually about 5-500 mg per
dosage unit formulation, specifically about 5-100 mg for an injection and
about 10-250 mg for other formulations. Each of the compositions
mentioned above may contain other active element as long as no
unfavorable interaction occurs with the antibody upon blending them.

[0153]Although a dosage of a pharmaceutical agent as a
prophylactic/therapeutic agent comprising the antibody of the invention
differs depending on the subject of administration, the target disease,
the symptom, the administration route and the like, when it is used, for
example, for treating adult type 2 diabetes, the antibody of the
invention is preferably administered as an intravenous injection for
usually about 0.01-20 mg/kg (weight), preferably about 0.1-10 mg/kg
(weight), more preferably about 0.1-5 mg/kg (weight) at a time for about
1-5 times a day, preferably for about 1-3 times a day. Furthermore, when
it is used for treating adult obesity, the antibody of the invention is
preferably administered as an intravenous injection for usually about
0.01-20 mg/kg (weight), preferably about 0.1-10 mg/kg (weight), more
preferably about 0.1-5 mg/kg (weight) at a time for about 1-5 times a
day, preferably for about 1-3 times a day. These dosages may similarly
apply to administration for the prevention of type 2 diabetes and
obesity, or for other parenteral (e.g., subcutaneous) or oral
administration. When the symptom is particularly severe, the dosage may
be increased accordingly.

[0190]Hereinafter, the present invention will be described more
specifically by means of examples, although the present invention should
not be limited thereto.

Example 1

Materials and Methods

[0191]1. Chemotaxis Assay

[0192]Mouse skeletal muscle-derived myoblast cell line C2C12 (obtained
from the American Type Culture Collection (ATCC)) and human monocytic
leukemia cell line THP-1 (obtained from the Health Science Research
Resources Bank, HSRRB), each obtained from an official cell bank, were
each cultured in a Dulbecco's modifies Eagle's medium (DMEM) containing
10% fetal bovine serum or an RPMI-1640 medium containing 10% fetal bovine
serum, and the cells at the log growth phase were used for the
experiments. 2×105 C2C12 cells were seeded on a 100-mm plate,
cultured at 37° C. overnight, added with forskolin (Sigma) to a
final concentration of 20 μM and cultured at 37° C. for another
two days. 106 THP-1 cells were seeded on a 100-mm plate,
simultaneously added with forskolin (Sigma) to a final concentration of
20 μM and cultured at 37° C. for two days. The forskolin is a
reagent that increases the intracellular cAMP concentration, which
activates A kinase and the transcription factors downstream therefrom. A
forskolin-stimulated THP-1 cell line is widely used for immunological
research as a cell line that has a similar nature to that of an activated
macrophage.

[0193]550 μl each of mouse CXCL14 (R&D Systems) and human CXCL14
(Peprotec) diluted with a chemotaxis buffer [0.1% Fatty acid-free BSA
(Sigma)-20 mM HEPES pH 8.0 (Invitrogen)-DMEM] to a final concentration of
100 nM were added to 24-well plates containing the C2C12 cells and the
THP-1 cells, respectively. For neutralization experiments with
antibodies, sheep anti-mouse CXCL14 polyclonal antibody (AF730, R&D
Systems) or rat anti-mouse CXCL14 monoclonal antibody (MAB730, R&D
Systems) was added to the chemotaxis buffer containing CXCL14 to a final
concentration of 10 μg/ml. Next, Chemotaxicell microchambers (Kurabo)
with pore sizes of 8 μm and 5 μm for the C2C12 cells and the THP-1
cells, respectively were left to stand still on the reaction solutions.
The forskolin-stimulated C2C12 cells or the forskolin-stimulated THP-1
cells were washed once in a chemotaxis buffer to remove the fetal bovine
serum. Thereafter, 2×105 cells/200 μL were layered on the
Chemotaxicell microchambers. The resulting 24-well plates were cultured
at 37° C. for 6 hours (C2C12 cells) or 2 hours (THP-1 cells). At
the end of the reaction, the culture solutions as the upper layer in the
microchambers were immediately removed and cells attached to the bottom
of the microchamber were fixed and stained with Diff-Quik (International
Reagents). Cells remaining at the upper layer of the microchambers were
removed with a cotton bud. The membranes of the microchambers were
removed and mounted on glass slides to count the number cells that
migrated around the pores under an optical microscope (100×). The
measurements were carried out with n=4 or more subjects for each sample
and StatView-J5.0 (SAS Institute) was run for statistical processing.

[0194]2. Western Blot Analysis

[0195]The C2C12 myoblast cell line was cultured in a DMEM medium
containing 5% horse serum for four days to induce muscle differentiation.
Following cultivation in a serum-free DMEM medium at 37° C. for 16
hours, the myocytes were treated with 100 nM mouse CXCL14 (R&D Systems)
for an hour with further addition of 10 nM insulin (Sigma) for
stimulation at 37° C. for 10 minutes. For neutralization
experiments with antibodies, sheep anti-mouse CXCL14 polyclonal antibody
(AF730, R&D System) was added to a pre-treated medium containing CXCL14
to a final concentration of 10 μg/ml. As a negative control, a medium
containing purified IgG from a non-immunized sheep was used. These cells
were dissolved, and 20 μg of the protein mixture was subjected to
SDS-polyacrylamide gel electrophoresis, followed by western blot analysis
with anti-Akt-pSer473 antibody and anti-Akt antibody (Cell signaling).
Akt is serine/threonine kinase also known as protein kinase B (PKB) which
is activated in PI3 kinase pathway and involved in various phenomena such
as insulin metabolism.

[0196]<Results and Discussion>

[0197]The chemotaxis, i.e., a typical biological activity, of CXCL14 was
analyzed by an in vitro assay using Chemotaxicell microchamber. As has
been reported previously (Nara N et al., J. Biol. Chem., vol. 282, pp.
30794-30803, 2007), muscle-differentiated C2C12 cells react with CXCL14.
Hence, the cells were subjected to a chemotaxis assay following
stimulation of the C2C12 cells with forskolin, a general differentiation
inducing reagent. As a result, cell attraction by mouse CXCL14 was
detected (FIG. 1). A similar activity was also detected for human CXCL14
(FIG. 1). Since only two residues among the 77 amino acids consisting
their polypeptides were different between the mouse CXCL14 and human
CXCL14 (see SEQ ID NOS: 1 and 2), the receptors expressed in the C2C12
cells were expected to have cross-reacted with the human CXCL14. Next,
mouse CXCL14-specific antibodies were added to the chemotaxis assay
solutions, where attraction of C2C12 cells by mouse CXCL14 was inhibited
by 61% and 54% with a polyclonal antibody AF730 and a monoclonal antibody
MAB730, respectively (FIG. 2). Subsequently, the forskolin-stimulated
THP-1 cells were used for the same chemotaxis assay, where cell
attraction by CXCL14 was inhibited by 79% by addition of anti-mouse
CXCL14 polyclonal antibody AF730 (FIG. 3). These results prove that
addition of anti-CXCL14-specific antibody can neutralize biological
activity of CXCL14 on two types of cells as target cells of CXCL14
(skeletal myocytes and activated macrophages) involved in induction of
obese insulin resistance. In particular, since activated macrophages that
accumulate in visceral fat are known as a major aggravating factor of
obese diabetes, the anti-CXCL14 antibody capable of suppressing the
chemotaxis of the activated macrophages was found to be useful as an
antidiabetic agent. When an anti-mouse CXCL14 monoclonal antibody MAB730
was added to an assay system of THP-1 cell, chemotaxis of THP-1 cells to
human CXCL14, to the contrary, increased (FIG. 4). The mechanism of this
phenomenon is yet unknown but it indicates that the CXCL14-specific
monoclonal antibody possibly controls the biological activity of CXCL14
in both negative and positive manners.

[0198]In a differentiated C2C12 myocytes, phosphorylation (activated
state) of the serine residue at position 473 of the Akt kinase due to
insulin stimulation is known to be partially inhibited by pre-treatment
with CXCL14 (Nara N et al., J. Biol. Chem., vol. 282, pp. 30794-30803,
2007). Thus, we studied whether an anti-CXCL14 antibody can neutralize
the insulin signal inhibitory activity by CXCL14. In the presence of a
control antibody, the band of phosphorylated Akt weakened by
pre-treatment with mouse CXCL14 whereas the band of phosphorylated Akt
was still strong in the presence of anti-mouse CXCL14 polyclonal antibody
AF730 (FIG. 5). This result indicates that addition of
anti-CXCL14-specific antibody can neutralize the insulin signal
inhibitory activity by CXCL14 in the myocytes. Myocytes are major tissue
that is responsible for the sugar level regulation by insulin. Since a
CXCL14 expression level in the myocytes increases along with obesity, a
partial inhibition of insulin signal by CXCL14 also seemed to contribute
to systemic insulin resistance by obesity. The anti-CXCL14 antibody was
expected to be effective to a secondary activity of CXCL14 associated
with onset of diabetes.

[0199]In studies using forskolin-stimulated C2C12 cells, cell attraction
by mouse CXCL14 was pertussis toxin-sensitive which was similar to the
response to a different type of chemokine CXCL12. Therefore, a CXCL14
receptor appears to be a trimeric G protein-coupled seven-transmembrane
protein, although no CXCL14 receptor gene has been isolated or identified
so far in any species including human and mouse. The anti-CXCL14-specific
antibody is not only useful as a tool for alleviating pre-diabetic state
caused by CXCL14 excessively produced by obesity but also expected to be
useful in clarifying the yet unknown physiological functions and receptor
structure of CXCL14.

INDUSTRIAL APPLICABILITY

[0200]The present invention provides an antibody that recognizes a
compound that suppresses or inhibits BRAK (CXCL14) functions, more
particularly an antibody (preferably, a human monoclonal antibody) that
specifically recognizes BRAK. The invention also provides a hybridoma
cell that produces the antibody and a method for producing the antibody.
In addition, the present invention provides, as use of the compound
mentioned above (particularly, an anti-BRAK antibody), a diagnostic agent
for type 2 diabetes and obesity, an agent for preventing and/or treating
type 2 diabetes and obesity, an agent that decreases the number of
macrophages in white adipose tissue, or an agent that ameliorates insulin
resistance, a method for preventing and/or treating type 2 diabetes and
obesity, a method for decreasing the number of macrophages in white
adipose tissue and a method for ameliorating insulin resistance.

[0201]The compound mentioned above (particularly, an anti-BRAK antibody)
is extremely useful in that it can be used as a novel therapeutic agent
for type 2 diabetes as well as an agent for diagnosing the risk of type 2
diabetes and obesity, and further as a possible agent for relieving
obesity.